1. Field of the Invention
This invention relates to a novel promoter for gene expression and uses thereof. More particularly, this invention relates to nucleic acid sequences encoding a functional promoter for the VDUP1 gene that correspond in structure and/or properties to the native genomic form of this promoter, and the use of such nucleic acid sequences for heterologous nucleic acid sequence expression.
2. Background
The VDUP1 gene (designated for Vitamin D3 UP-regulated 1) was first described as a gene strongly induced by vitamin D3 in human HL-60 cells. See, e.g., Chen and DeLuca, Biochem. Biophys. Acta, 1219:26-32 (1994). The exposure of HL-60 cells to vitamin D3 leads to cell differentiation into monocytes or macrophages. In that study, VDUP1 was isolated from a cDNA library by differential hybridization of mRNA from cells treated or untreated with 1,25-dihydroxyvitamin D3. Induction of VDUP1 by vitamin D3 was shown to occur even in the presence of the protein synthesis inhibitor, cyclohexamide. Further, exposure to cyclohexamide alone could induce VDUP1 several fold. More recently, it has been determined that VDUP1 is down-regulated in rat tumors generated by N-methyl-N-nitrosurea induction. See e.g., Young, et al., Mol. Carcinog., 4:251-60 (1996); Yang, et al., Breast Cancer Res. Treat., 48:33-44 (1998). No correlation between these two modulatory effects on VDUP1 expression has been characterized. Clues to the biological function of the VDUP1 gene product were recently described in a paper by Nishiyama, et al. J. Biol. Chem., 274:21645-50(1999). Protein binding studies in the yeast two-hybrid system, identified the VDUP1 gene product as a protein that specifically binds to the disulfide reducing protein, thioredoxin (TRX). These provide evidence that VDUP1 protein may only bind to reduced TRX and perhaps inhibits its reducing potential. In addition, overexpression of VDUP1 inhibits the expression of TRX. In HL-60 cells treated with vitamin D3, the induction of VDUP1, in agreement with the results of Chen and DeLuca, Biochem. Biophys. Acta, 1219:26-32 (1994), correlates with a significant decrease in the expression of TRX. TRX over-expression has been correlated with growth promotion. The biological significance of this direct effect of VDUP1 on TRX expression and redox function may be to modulate oxidation-reduction regulation of cellular factors and processes involved in cell growth and differentiation.
Antibodies represent one of the best therapeutic candidates for the treatment of disease due to their inherent specificity for their target molecule. However, a major drawback to their effective clinical use is the fact that they are bio-molecules and must be synthesized by expression from transfected cells in culture or transgenic plants or animals. The same is true for any reactive bio-molecule, such as a growth factor or enzyme, that may have utility in research or clinical application. To date, the majority of bio-molecules now produced are generated from cultured cells. Systems have been developed to enhance the overall output of protein production from cells, including promoter selection, gene targeting, gene amplification, highly selective transfection markers, fluorescence activated cell sorting, and medium supplementation. Each of these have shown some degree of success in enhancing gene expression of the desired molecule. In regard to promoter selection, the majority of expression vectors used for this application employ viral promoters, such as cytomegalovirus (CMV) or SV40. The CMV promoter in particular is often utilized primarily due to its strong constitutive expression in a variety of cell types and its propensity for up-regulation by exogenous agents and cellular stress. During standard cell culture for the production of bio-molecules, expression of recombinant protein occurs in a fairly linear fashion throughout the culture period, typically 10-14 days. However, noticeable enhancement of production usually occurs in late culture. Typically, expression of the CMV promoter is also frequently down-regulated following stable integration into cellular genomes, despite exhibiting strong expression in transient transfection experiments (M. Stiaski, Gene Expression Systems, 211-233, (J. M Fernandez and J. P Hoeffler ed., Academic Press 1999)). Thus there is a need for a strong constitutive promoter whose regulation can be enhanced during all stages of prolonged cell culture and upon genomic integration.
This invention relates to a substantially purified nucleic acid molecule comprising a VDUP1 promoter region, whose expression is induced to maximal levels as a result of prolonged cell culture. This invention also relates to a nucleic acid molecule having a sequence consisting of: (a) a nucleic acid sequence substantially similar to that of SEQ ID NO:1, or a fragment thereof, which exhibits promoter activity; (b) a nucleic acid sequence substantially complementary to the nucleic acid sequence of (a) or an active fragment thereof; or (c) a nucleic acid sequence that hybridizes to the nucleic acid sequences of (a) or (b) or fragments thereof under high stringency conditions. In another aspect, the invention relates to expression vectors comprising the aforementioned nucleic acid sequences and host cells transformed with these expression vectors.
The invention also relates to methods for detecting test agents which modulate the expression of the VDUP1 promoter described above comprising contacting a host cell transformed with an expression vector comprising the VDUP1 promoter DNA sequence operably linked to a reporter nucleic acid sequence with the test agent and comparing the level of transcription of the reporter nucleic acid sequence product produced in the presence of the test agent to the level of transcription produced in its absence.
In one embodiment of this aspect of the invention there are provided methods of screening for test compounds or factors that modulate the activity of the VDUP1 promoter by: (a) contacting host cells transformed with a nucleic acid molecule containing the VDUP1 promoter disclosed herein operably linked to a reporter nucleic acid sequence with a test medium containing the test compound under conditions which allow for expression of the reporter nucleic acid sequence; (b) measuring the expression of the reporter nucleic acid sequence in the presence of the test medium; (c) contacting host cells with a control medium which does not contain the test compound but is otherwise identical to the test medium in (a), under conditions identical to those used in (a); (d) measuring the expression of reporter nucleic acid sequence in the presence of the control medium; and (e) correlating any difference in expression between (b) and (d) to the ability of the test compound to regulate the activity of the VDUP1 promoter.
The invention also relates to methods of expressing a heterologous nucleic acid sequence in a mammal comprising introducing into mammalian cells a vector comprising the nucleic acid sequence encoding a VDUP1 promoter operably linked to a nucleic acid sequence encoding a heterologous protein, polypeptide, hormone, ribozyme or antisense RNA, whereby the heterologous protein, polypeptide hormone, ribozyme or antisense RNA is expressed.
The invention also relates to transgenic or chimeric animals whose cells express a heterologous nucleic acid sequence under the transcriptional control of a VDUP1 promoter.